Transglutaminases (EC 2.3.2.13: TG) are protein cross-linking enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxy-amide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds such as peptides, proteins, and similar compounds may function as acyl acceptors with the subsequent formation of monosubstituted γ-amides of peptide bound glutamic acid. When the ε-amino group of a lysine residue in a peptide or polypeptide chain serves as the acyl acceptor, the transglutaminases form intramolecular or intermolecular γ-glutamyl-ε-lysyl crosslinks.
The crosslinking activity of transglutaminases has been shown to be useful for a variety of industrial purposes, including in the field of food processing, such as processing of raw fish meat paste, tofu noodles, confectionery/bread, food adhesives, sheet-like meat food, yogurt, jelly, cheesegelling of proteins, improving baking quality of flour, improving taste and texture of food proteins, as well as casein finishing in leather processing, etc. Transglutaminases have also been employed in the production of thermally stable materials such as microcapsules, carriers of immobilized enzymes and the like.
A wide array of transglutaminases have been identified and characterized from a number of animal species and a few plant species. The most widely used animal derived transglutaminase, factor XIIIa, is a Ca2+-dependent multi-subunit enzyme. Factor XIIIa is a product-inhibited enzyme, which means the activity of the enzyme is inhibited by the product synthesized after the enzymatic reaction. Such a property is a disadvantage for many industrial applications and for obtaining product of the enzymatic reaction. A Ca2+-dependent transglutaminase from the slime mold Physarum polycephalum has also been described in Klein et al., (1992). However, only few microbial transglutaminases have been disclosed, e.g., from the species Streptoverticillium lividans, Streptoverticillium mobaraense, Streptoverticillium cinnamoneum, and Streptoverticillium griseocarneum (in U.S. Pat. No. 5,156,956) and from the species contemplated to be Streptomyces lavendulae (in U.S. Pat. No. 5,252,469, and U.S. Pat. No. 5,156,956). Bacterial transglutaminases which do not require the presence of calcium for their activity are usually identified and tested by using a conventional enzyme assay in which hydroxylamine is converted to hydroxamic acid (Folk, J. E. & Cole, P. W. (1966)).
Biological agents such as transglutaminases have limitations in that they cross-link only a limited number of very specific compounds, i.e. they are very as substrate-specific. Moreover, despite some industrial applications, biological agents have not been used as cross-linking agents for preparing antigens or in other immunological applications. Most known cross-linking biological agents such as enzymes have not been considered desirable for immunological applications due to problems such as the lack of an adequate quantity of the enzymes, high cost, difficulty in purification, and the like. For example, the cross-linking biological agent, microbial transglutaminase, has been purified mainly from culture medium (JP-B-6-65280, Agric. Biol. Chem., vol. 69, no. 10, pp. 1301–1308). Microbial transglutaminases purified from crude lysate, culture medium, or batch fermentation may not be suitable for vaccine development due to contamination by toxic compounds or other cellular proteins or components which may induce undesirable cross-reactive antibodies.
One approach to prepare transglutaminase has been to use recombinant DNA techniques to produce bacterial strains that produce recombinant transglutaminases. For example, the Streptoverticillium mobaraense transglutaminase gene has been cloned for expression in Escherichia coli, Streptomyces lividans, and Saccharomyces cerevisiae (Washizu et al., Tahekana et al., and EP-A-0 481 504). However, even the most successful of these approaches (Washizu et al.) resulted in a production yield much lower than the yield in the wildtype Streptomyces mobaraense strain. Thus, none of the efforts to overproduce the S. mobaraense enzyme have been successful, despite utilization of a number of different approaches such as chemical synthesis of a codon-optimized gene and its subsequent expression (as a cleavable heterologous signal peptide fusion to the mature transglutaminase) to the periplasm of E. coli or S. cerevisiae, expression as a fusion protein to pro-transglutaminase in S. cerevisiae, and traditional isolation and expression of the natural transglutaminase from wildtype S. mobaraense. 
Furthermore, protein cross-linking reactions by transglutaminase have the following problems. Since transglutaminase is an enzyme forming an intramolecular or intermolecular bridge as a result of the acyl rearrangement reaction, some proteins or peptides cannot serve as substrates for the enzyme due to an insufficient number of glutamine residues or lysine residues. For example, albumin proteins cannot be used as the substrate for transglutaminase in its native form despite the presence of intrinsic glutamine and lysine residues.
It would, therefore, be desirable to engineer a recombinant transglutaminase with a broad substrate specificity that can be efficiently and effectively be purified with a large yield. Such a transglutaminase could be used in antigen preparation, vaccine development, immunotherapy, and medical diagnostic applications. It further would be desirable to develop a reproducible transglutaminase purification procedure so as to meet the FDA purity standard required for the use of transglutaminase in a vaccine and/or diagnostic detection kit.